Treatment of pressure ulcers in patients with declining renal function using arginine, glutamine and ß-hydroxy-ß-methylbutyrate.

The aim of this study is to examine the efficacy on healing pressure ulcers (PU) of using a supplement combination containing arginine, glutamine and ß-hydroxy-ß-methylbutyrate, which was given to two elderly patients with renal dysfunction. The PU was surgically opened, decompressed and treated by drugs. A half quantity of the defined dose of the supplement combination, with an enteral nutrition product, was administered to the patients twice a day. This combination improved the PUs, with no effect on renal function. This novel finding may provide a nutritional rationale of arginine, glutamine and ß-hydroxy-ß-methylbutyrate for PUs associated with renal dysfunction.

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation.

The influence of untranslated 5' sequences on c-myc expression was compared by measuring the translational efficiencies of mRNAs which contain leaders derived from exon 1 or intron 1 of the human c-myc gene. Expression plasmids were constructed and introduced into COS cells, and the levels of c-myc mRNA and protein were examined. Our results show that mRNAs transcribed from constructs containing exon 1 or intron 1, which have different folding potential, are translated with approximately equal efficiencies. This suggests that the translation of c-myc mRNA is not controlled by secondary structure alone. In addition, we observed that transcripts in which exon 1 was deleted are not translated more efficiently, but are present at a higher steady-state level. Thus, this example provides evidence for possible control at the transcriptional level. Finally, since the c-myc product was produced in each of our test systems, the results suggest that this protein does not regulate its own transcription or translation via a specific interaction with c-myc exon 1 alone.

Genotoxicity (mitotic recombination) of the cancer chemotherapeutic agents cisplatin and cytosine arabinoside in Aspergillus nidulans.

Cisplatin (cis-diamminedichloroplatinum, cis-DDP) and cytosine arabinoside (ara-C) are anticancer drugs used in the treatment of human cancer. The two chemotherapeutic drugs were tested in current research for their recombinogenic potential in diploid cells of Aspergillus nidulans. Non-cytotoxic concentrations of ara-C (0.4 and 0.8 microM) and cis-DDP (1.5, 3.0 and 6.0 microM) were strong recombinagens in A. nidulans UT448//A757 diploid strain, which induced homozygosis of recessive genetic markers, previously present in heterozygous condition. Drugs significantly increased homozygosity index (HI) values for five nutritional genetic markers when compared with those determined in the absence of anticancer drugs. Since mitotic recombination is a mechanism leading to malignant growth through loss of heterozygosity at tumor-suppressor loci, ara-C and cis-DDP may be characterized as secondary promoters of malignant neoplasia in diagnosed cancer patients, after chemotherapy treatment.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Implications of protein conformational diversity for binding and development of new biological active compounds.

The new generation of biologically active compounds developed during the 20(th) century relied on knowledge of enzymology and protein structure, and were based initially, on the understanding that protein-protein and small molecule-protein interactions occurred through a lock-and-key mechanism. Later, evidence suggested that this mechanism was usually followed by a conformational change, known as induced fit. Recent studies on protein dynamics, mainly by nuclear magnetic resonance (NMR) relaxation measurements, have shown that proteins are not structured in a unique conformation. Rather, they frequently have regions of conformational diversity. In the present review we will discuss a novel view of binding, put forward in by several research groups in the last 5 to 10 years. In the free state, protein regions displaying conformational diversity exhibit equilibria among pre-existing conformations. In the presence of a ligand, one of these conformations is stabilized, so that the ligand does not need to induce a new conformation. Upon ligand binding there is a population shift toward the bound conformational state. Conformational diversity of binding sites of several proteins has been measured and has important practical as well as thermodynamical consequences: binding sites can be mapped without prior knowledge of the ligand and also evolution of binding sites depends mostly on the free state, occurring at least partially independently of the ligand.

Design and performance of a compact scanning transmission X-ray microscope at the Photon Factory.

We present a new compact instrument designed for scanning transmission X-ray microscopy. It has piezo-driven linear stages, making it small and light. Optical components from the virtual source point to the detector are located on a single optical table, resulting in a portable instrument that can be operated at a general-purpose spectroscopy beamline without requiring any major reconstruction. Careful consideration has been given to solving the vibration problem common to high-resolution microscopy, so as not to affect the spatial resolution determined by the Fresnel zone plate. Results on bacteriogenic iron oxides, single particle aerosols, and rare-earth permanent magnets are presented as examples of its performance under diverse applications.

Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Covalent modification of all members of human cullin family proteins by NEDD8.

Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.

Identification of specific regions of the human endothelin-B receptor required for high affinity binding with endothelin-3.

To investigate the endothelin-3 (ET-3) binding region of the endothelin-B (ETB) receptor, we have transiently produced various chimeric endothelin receptors in transfected Chinese hamster ovary cells. Using 125I-ET-1 as the radioactive ligand in the displacement experiment, the replacement of both the second and third extracellular regions including the flanking transmembranes of the ETB receptor with the corresponding domains of the endothelin-A (ETA) receptor, increased the apparent Ki value for ET-3 from 5 x 10(-11) M to 10(-8) M. The affinity of this chimeric receptor, ETB-BC, for ET-3 was about two orders lower than ETB yet one order higher than ETA. Previously we have reported the involvement of Lys-140 located in the C-terminus of the second transmembrane region of the ETA receptor for ET-1 binding (Eur. J. Biochem., 220, 37-43, 1994). To assess the importance of the corresponding Lys-161 of the ETB receptor in ET-3 binding, we have replaced it with Ile in the ETB receptor. The mutant receptor had a 5.6-fold reduction in its affinity for ET-3, but its affinity for ET-1 remained similar. These results demonstrate that Lys-161 of the receptor is important for high affinity binding with ET-3 which, in part, confers the non-selective binding characteristics of the ETB receptor for ET isopeptides.

Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi.

LuxO is the central regulator integrating the quorum sensing signals controlling autoinduction of luminescence in Vibrio harveyi. We have previously purified to homogeneity a new lux regulator, LuxT, that binds to the luxO promoter. Based on the sequence of the tryptic peptides of LuxT, degenerate oligonucleotides were designed for PCR of the genomic DNA. A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. The recombinant and native LuxT gave the same footprint binding between 117 and 149 bp upstream from the luxO initiation codon. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT was a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.

The LuxR regulator protein controls synthesis of polyhydroxybutyrate in Vibrio harveyi.

The LuxR regulatory protein of Vibrio harveyi has been shown to control synthesis of polyhydroxybutyrate (PHB) as well as luminescence so as to occur at high cell density, suggesting that it is a general regulatory protein. Mutants defective in the production of LuxR (D1, D34, and MR1130) were found to be missing PHB, whose synthesis could be restored by complementation with luxR. Triparental mating with a V. harveyi genomic library revealed the presence of three genomic clones (G1, G2 and G3) that could also restore PHB synthesis and luminescence to cells which express low levels of luxR (D1 and D34) but not to luxR- cells (MR1130) suggesting that luxR expression was being stimulated. Analyses of luxR mRNA levels by mRNA dot blot hybridization and by primer extension confirmed that luxR mRNA levels were increased 4 to 7-fold in the D1 and D34 cells by the G1, G2 and G3 fragments and show that expression of a single genomic copy of luxR is sufficient to restore synthesis of PHB. The results demonstrate that V. harveyi LuxR controls the induction of a process not intimately involved in the bioluminescence system and clearly distinguishes its role in V. harveyi from that of LuxR from Vibrio (Photobacterium) fischeri, which has only been associated with regulation of light emission.

The gene convergent to luxG in Vibrio fischeri codes for a protein related in sequence to RibG and deoxycytidylate deaminase.

The nucleotide sequence of a convergent gene with the same bidirectional transcriptional terminator as the Vibrio fischeri lux operon has been determined. This gene codes for a polypeptide of 147 amino acids which is related in sequence to the polypeptide coded by the first gene (ribG) of the rib operon of Bacillus subtilis as well as deoxycytidylate deaminase of T4 bacteriophage and Saccharomyces cerevisiae. These results raise the possibility of a linkage between the regulation of the lux genes and riboflavin synthesis in Vibrio fischeri.

Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli.

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.

Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.

Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.

Effects of SR-spirochete infection on Drosophila melanogaster carrying intersex genes.

Three sex-transforming genes (ix, tra-D, and dsx) in D. melanogaster were examined with respect to possible interactions with the NSR strain SR-spirochetes. The SR-spirochetes exerted a lethal effect on XY but not on XX individuals, regardless of whether they were phenotypically males, intersexes, or females. These results, taken together with those reported by Sakaguchi and Poulson (1963)on tra and 2X3A intersexes, both of which are resistant to the androcidal action of SR-spirochetes, support the interpretation that male susceptibility is a consequence of the single X condition.

Identification of a domain of ETA receptor required for ligand binding.

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ-123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B-loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ-123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B-loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB-based chimeric receptor with a sensitivity to BQ-123. These observations suggest that the B-loop domain of the ETA receptor is involved in ligand binding.

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle.

We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.